EBioMedicine 73 (2021) 103622

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EBioMedicine
journal homepage: www.elsevier.com/locate/ebiom

Research paper

Severe COVID-19 is characterized by the co-occurrence of moderate

cytokine inflammation and severe monocyte dysregulation
Benjamin Bonneta,b, Justine Cosmea, Claire Dupuisc, Elisabeth Coupezc, Mireille Addac,
Laure Calvetc, Laurie Fabrea, Pierre Saint-Sardosd, Marine Bereiziatc, Magali Vidale,
Henri Laurichessee, Bertrand Souweinec, Bertrand Evrarda,b,*
a
Service d'Immunologie, CHU Gabriel-Montpied, Clermont-Ferrand, France
b
Laboratoire d’Immunologie, ECREIN, UMR1019 UNH, UFR Me
decine de Clermont-Ferrand, Universite
Clermont Auvergne, Clermont-Ferrand, France
c
Service de Me
decine Intensive et Re
animation, CHU Gabriel-Montpied, Clermont-Ferrand, France
d
Laboratoire de Bacte
riologie, CHU Gabriel-Montpied, Clermont-Ferrand, France
e
Service de Maladies Infectieuses et Tropicales, CHU Gabriel-Montpied, Clermont-Ferrand, France

A R T I C L E I N F O A B S T R A C T

Article History: Background: SARS-CoV-2 has been responsible for considerable mortality worldwide, owing in particular to
Received 27 February 2021 pulmonary failures such as ARDS, but also to other visceral failures and secondary infections. Recent progress
Revised 10 September 2021 in the characterization of the immunological mechanisms that result in severe organ injury led to the emer-
Accepted 28 September 2021
gence of two successive hypotheses simultaneously tested here: hyperinflammation with cytokine storm
Available online xxx
syndrome or dysregulation of protective immunity resulting in immunosuppression and unrestrained viral
dissemination.
Keywords:
Methods: In a prospective observational monocentric study of 134 patients, we analysed a panel of plasma
SARS-CoV-2
Intensive care unit
inflammatory and anti-inflammatory cytokines and measured monocyte dysregulation via their membrane
Inflammation expression of HLA-DR. We first compared the results of patients with moderate forms hospitalized in an
Monocyte dysregulation infectious disease unit with those of patients with severe forms hospitalized in an intensive care unit. In the
Immunosuppression latter group of patients, we then analysed the differences between the surviving and non-surviving groups
HLA-DR and between the groups with or without secondary infections.
Secondary infection Findings: Higher blood IL-6 levels, lower quantitative expression of HLA-DR on blood monocytes and higher
IL-6/mHLA-DR ratios were statistically associated with the risk of severe forms of the disease and among the
latter with death and the early onset of secondary infections.
Interpretation: The unique immunological profile in patients with severe COVID-19 corresponds to a moder-
ate cytokine inflammation associated with severe monocyte dysregulation. Individuals with major CSS were
rare in our cohort of hospitalized patients, especially since the use of corticosteroids, but formed a very
severe subgroup of the disease.
Funding: None.
© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction complications of the acute respiratory distress syndrome (ARDS)

Since the detection of severe acute respiratory syndrome corona-
virus 2 (SARS-CoV-2) in Wuhan in 2019, the disease has spread very
rapidly to become a global pandemic currently responsible for more
than 4.5 million deaths. About 15% of coronavirus disease 19 (COVID-
19) patients over 60 years of age require hospitalization and 5%
require intensive care unit (ICU) admission [1,2]. ICU mortality rate is
high, ranging from 16 to 57%, and is mainly related to pulmonary
* Corresponding author.
E-mail address: bevrard@chu-clermontferrand.fr (B. Evrard).
[3 7]. Multiple organ failure (in 12 to 29% of cases) and secondary
infections (in 10 to 57% of cases) can also frequently occur [3,8,9].
Understanding the pathophysiology of severe COVID-19 forms,
especially of respiratory failure, is henceforth critical for determining
the best management and treatment strategies. Recent progress in
the characterization of the immunological mechanisms that result in
organ injury, with particular regard to the determinants of severity,
has shown the role of hyperinflammation, in particular elevated lev-
els of proinflammatory cytokines or chemokines associated with
anomalies of adaptive and innate immunity through disruption of
the lymphoid and myeloid lineages [10 12].

https://doi.org/10.1016/j.ebiom.2021.103622
2352-3964/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
2 B. Bonnet et al. / EBioMedicine 73 (2021) 103622
Research in Context
Evidence before this study
SARS-CoV-2 infections are associated with significant mortality,
mainly owing to lung damage such as ARDS, but also to damage
to other organs or to secondary infections. Two opposing path-
ophysiological hypotheses have been successively put forward
to explain these severe forms. In one, COVID-19 is considered
to be a hyperinflammatory disease with cytokine release syn-
dromes causing deleterious infiltration of immune cells into the
lung. In the other, it is rather a disease with a complex deregu-
lation of the immune system leading to uncontrolled viral
spread and thus ARDS. Few studies have described the immu-
nological characteristics of patients on both sides of the
immune response simultaneously and did not provide a
detailed description of the patient profile based on the severity
of the disease or on the mortality induced. Furthermore, they
did not at all describe whether these markers were associated
or not with the occurrence of secondary infections. All these
elements would however be necessary to guide therapeutic
choices in a personalized medicine approach that would adapt
treatment to the inflammatory or immunocompromised profile
of the patients.
Added value of this study
Our data provide new evidence that patients with severe forms
of COVID-19 most often exhibit moderate cytokine inflamma-
tion concurrently with severe monocytic dysregulation. We
show that a quantitative decrease in mHLA-DR membrane
expression and an increase in the IL-6/mHLA-DR ratio were
associated with severe forms of the disease, the degree of pul-
monary involvement, and the risk of death and onset of early
infections. In addition, we show that patients with a major
cytokine storm were rare in our cohort of hospitalized patients,
especially since the systematic use of corticosteroids, but corre-
sponded to a subgroup of patients with a very poor prognosis.
Implications of all the available evidence
The mHLA-DR expression and the IL-6/mHLA-DR ratio could be
used in a routine care approach to identify patients at risk for
severe forms, death and early secondary infections. This could
make it possible to better adapt the therapies implemented in a
personalized medicine approach, using treatments that target
cytokine inflammation or, depending on the patient's profile,
immunostimulant treatments.
The first related studies posed the hypothesis of the cytokine
storm syndrome (CSS), suggesting that the increase in pro-inflamma-
tory cytokines (IL-2, IL-6, IL-7, IP-10, TNFa. . .), which is marked in
ICU patients and associated with impaired interferon type 1 response,
induced the infiltration of inflammatory cells in the lung and thus
injury to the organ [3,12 14]. However, this hypothesis has recently
been called into question, in particular by comparing the level of
increase in cytokines with that observed in other pathological situa-
tions that induce CSS, such as sepsis and CAR-T cell infusion, and
those that do not, such as influenza [15 17].
A new explanation is gradually emerging that places emphasis on
more complex cellular immune dysregulation. Severe forms of
COVID-19 are associated with lymphopenia, including decreased
numbers of circulating T, B, and NK cells, exhibit activation of CD4 T
cells and decrease regulatory T cells associated with a skewing of
CD8+ T cells towards a terminally differentiated/senescent phenotype
[18 21]. Deep analysis also revealed drastic changes within the mye-
loid cell compartments during severe COVID-19. With regard to neu-
trophils, multiple abnormalities have been described such as
increased neutrophil/lymphocyte ratio, evidence of emergency mye-
lopoiesis with release of immature neutrophils, transcriptional pro-
grams typical of dysfunction and immunosuppression, and lung
recruitment with NETosis [22 25]. Abnormalities of monocytes have
been recently characterized, where expansion and early activation of
classical monocytes (CD14+CD11c++HLA-DR++) in the blood are char-
acteristics of mild COVID-19 which decrease during the natural
course of the disease. In contrast, in severe forms the non-classical
monocyte subpopulation (CD14loCD16++) appears depleted, probably
due to its lung recruitment, and the intermediate (CD14+CD16+) and
classical subsets (CD14++CD16 ) become dysfunctional with the loss
of HLA-DR membrane expression [11,18 21].
Here we hypothesize that the two phenomena described above,
hyperinflammation or dysregulation of protective immunity, proba-
bly took place at the same time in the severe forms and were nega-
tively complementary rather than antagonistic. We therefore looked
for simple biological tools that are routinely available in our univer-
sity hospital centre to simultaneously test hyperinflammation, anti-
inflammatory cytokines and monocyte dysregulation by a panel of
plasma cytokines and by measuring the membrane expression of
human leukocyte antigen antigen D-related (HLA-DR) on the surface
of monocytes. We initially chose this last marker because it is now a
recognized diagnostic marker in the assessment of the severity of
sepsis-induced immunosuppression [22]. We first sought to see if
these markers were capable of distinguishing between mild forms
observed in the hospital infectious disease unit (IDU) and ICU severe
forms. Focusing on the severe forms, we then sought to assess the
potential of these markers to 1) determine possible differences in the
immune profiles between patients of the two successive COVID-19
waves in France, 2) identify patients according to whether they had
fatal evolution or not, and 3) predict the risk of secondary infections
in order to provide evidence of the real immunoparesis of the
immune system.
2. Methods
2.1. Patients and data collection
All patients with confirmed COVID-19 admitted to the IDU medi-
cal wards and ICU of the Gabriel-Montpied Teaching Hospital in Cler-
mont-Ferrand, France, from March to the end of November 2020
were eligible for enrolment. A total of 134 patients admitted to the
ICU were thus included in a preliminary prospective monocentric
observational study and followed up until ICU discharge and/or
death. In our hospital, as in the rest of France, we experienced two
distinctly separate waves of patient hospitalizations: the months of
March to June (03/08/2020 to 06/02/2020), a period called Wave 1,
during which 34 patients were enrolled, and from September to
November (09/14/2020 11/23/2020), a period called Wave 2, dur-
ing which 99 others were included. A single patient was hospitalized
in August between the two waves, but for the purpose of the study
was included in the Wave 1 group. We decided therefore a posteriori
to analyse the data of ICU patients based on their period of hospitali-
zation, especially since the results of the RECOVERY clinical trial, pub-
lished in the summer of 2020, showed the clinical benefit of
dexamethasone on 28-day mortality in patients requiring oxygen
support. This led to a change in practice in the management of ICU
COVID-19 patients with the introduction of dexamethasone as soon
as the patient required oxygen [23].
The immunoactivation/immunosuppression balance was moni-
tored in the patients at three different time points, on days 0-3, days
7-10 and at ICU discharge (endpoint).
 B. Bonnet et al. / EBioMedicine 73 (2021) 103622
ICU admission data, ie demographic characteristics, comorbidities,
severity score (SAPS II), nadir PaO2/FiO2 ratio, and laboratory find-
ings were also collected (Table 1). During their ICU stay, patients
were screened for nosocomial infections defined as microbiologi-
cally-proven pneumonia, complicated urinary tract infection (eg
prostatitis), bacteremia, septic shock, catheter-associated infections
and fungal infections. The survival status of included patients at dis-
charge from the ICU was recorded. In terms of sample size, due to our
pragmatic approach based on the interest in patients in daily care,
we initially wanted to test all patients hospitalized in the IDU and
ICU of our hospital without restrictive inclusion criteria to determine
whether the markers chosen were predictive of the risk of worsening.
However, this was not possible in practice for reasons of feasibility in
a pandemic context that led to obstacles such as shortage of stocks of
reagents and overwork of healthcare teams. Our aim at the outset
was to include all patients hospitalized in the IDU as reference cases
of mild-form COVID-19, which proved impossible because of organi-
zational constraints related to the influx of patients into the depart-
ment. We were therefore able to obtain only 10 IDU patients. To limit
the heterogeneity of the ICU patient group and to be able to compare
it with the IDU group, we decided to take into account for analysis
patients who entered the ICU directly and patients who entered the
ICU with prior admission to another department for a period <
3 days, but no patients from an ICU of another hospital. This reduced
our Wave 1 ICU group to a total of 25 patients but did not impact the
Wave 2 ICU group (see flow chart in Fig. 1).
2.2. Ethics
All patients or their relatives received fair and relevant information.
They gave written informed consent for the storage and research use of
residual blood from samples collected as part of routine care (IRB n°
20.03.20.56342 from CPP-Ile-de-France VI Groupe Hospitalier Pitie
-Sal-
^trie
pe re). Blood samples from voluntary healthy donors were collected
during the COVID-19 pandemic and then included as negative controls.
Their demographics are given in Table 1.
2.3. Analysis of HLA-DR expression on monocytes
Monocyte HLA-DR (mHLA-DR) expression, in numbers of antibod-
ies bound per monocyte (Ab/cell), was analysed at three time points,
on days 0-3, days 7-10 and at ICU discharge (endpoint). EDTA-blood
was drawn at the above time points after inclusion of patients in the
study. The blood was stored at 4-8°C and processed within 4 h after
withdrawal. The quantitative expression of mHLA-DR was deter-
mined with the anti-HLA-DR/anti-Monocyte QuantiBRITE assay (BD
Biosciences, San Jose, CA, USA). Briefly, whole blood (25 mL) was
stained with 10 mL of QuantiBRITE HLA DR/Monocyte mixture
(QuantiBRITE anti HLA DR PE (clone L243)/anti monocytes (CD14)
PerCP Cy5.5 (clone MfP9), Becton Dickinson San Jose, CA, USA) at
room temperature for 30 min in a dark chamber. Samples were then
lysed using the FACS Lysing solution (Becton Dickinson San Jose, CA,
USA) for 15 min. After a washing step, cells were acquired on a BD
LSR II cytometer (BD Biosciences, San Jose, CA, USA) and flow data
were analysed with FlowLogic software (version 7.3 software, Milte-
nyi Biotec, Germany). The total number of antibodies bound per
monocyte (Ab/cell), defined as total CD14+ cells, were quantified by
calibration with a standard curve determined with BD QuantiBRITE
phycoerythrin (PE) beads (BD Biosciences, San Jose, CA, USA) (see
Figure S1 for gating strategy). Antibodies, reagents and the respective
suppliers are shown in Supplemental Table 1.
2.4. Plasma cytokine analysis
Plasma was obtained through centrifugation of EDTA samples
within 4 h of phlebotomy at the indicated time points following
3
inclusion of patients in the study. Plasma was placed in aliquots and
stored at 20°C until use. Human pro-inflammatory cytometric bead
array (CBA) and human IFN-a flex kits (BD Biosciences, San Jose, CA,
USA) were used. The human pro-inflammatory cytokine kit simulta-
neously detects IL-6, IL-10 and CXCL8 cytokines in a single sample
whereas the IFN-a flex kit detects IFN-a. The assays were performed
according to the manufacturer's instructions. Samples and standards
were acquired on the BD LSR II cytometer (BD Biosciences, San Jose,
CA, USA) and the generated FSC files were analysed with FCAP Array
version 3.0 software.
Plasma IL-1Ra levels were measured with a commercial enzyme-
linked immunoassay (ELISA) kit (human IL-1Ra ELISA from Invitro-
gen, Villebon-sur-Yvette, France). Plasma samples were frozen and
stored for batch analysis. In brief, to determine IL-1Ra levels, samples
were thawed and 50 mL aliquots were incubated in microtitre wells
coated with anti-human IL-1Ra antibody. The wells were then
washed and detection achieved by adding biotin-conjugated anti-
human IL-1Ra antibody followed by incubation with streptavidin-
HRP, and finally by addition of horseradish peroxidase (HRP) sub-
strate solution. A coloured product formed in proportion to the
amount of human IL-1Ra present and absorbance was measured at
450 nm. Reagents and the respective suppliers are depicted in Sup-
plemental Table 1.
2.5. Statistics
Statistical analyses and graphical presentations were conducted
with GraphPad Prism version 8.0 software (Graph-Pad Software). The
population was expressed as numbers and percentages for categori-
cal variables. For quantitative variables, the results were expressed in
terms of mean § standard deviation in the Figures or mean and IC95
in the Tables. Categorical data were analysed with Chi-square test
with a 5% risk of type 1 error. Statistical analysis of continuous varia-
bles, ie the different severity groups, mild versus severe forms, survi-
vors versus non-survivors, was performed with one-way
Mann Whitney U test. For the longitudinal follow-up of patients,
statistical analysis from baseline was performed with a Wilcoxon
matched pairs test. Pearson’s correlation coefficient was used to
assess the relationship between the PaO2/FiO2 ratio and mHLA-DR
expression or IL-6 and mHLA-DR expression. Analyses were per-
formed with no correction for multiplicity. The receiver operating
characteristics (ROC) curve analysis was performed using the “pROC”
package on R software to identify the optimal cut-off values of base-
line and D7-10 mHLA-DR, IL-6 or IL-6/mHLA-DR*103 parameters for
prediction of fatal COVID-19 or secondary infections. The optimal
cut off points to predict the severity of COVID 19 were determined
by Youden's index using R software. Sensitivity, specificity and the
corresponding IC95 of optimal cut-off points were determined using
the “pROC” package.
2.6. Role of funders
None. The study was based on routine care and received no finan-
cial sponsorship.
3. Results
3.1. Cohort of consecutive patients
A total of 134 patients who were laboratory-confirmed to be
infected with SARS-CoV-2 in hospital were included. The immuno-
logical characteristics and their clinical and laboratory features were
compared between 124 severe cases admitted to the ICU and 10 mild
cases admitted to the IDU (Table 1). ICU patients were more often
men and were older than IDU patients. They also had a more severe
inflammatory profile induced by COVID-19 as evidenced by a much
 4
Table 1
Characteristics of patients admitted to the ICU or IDU for SARS-CoV-2 infection at the time of enrollment.

Total ICU (n = 124) ICU (n = 25) Wave 1 ICU (n = 99) Wave 2 P value Wave 1 vs IDU (n = 10) P value Total Healthy controls Reference range
Wave 2 ICU vs IDU (n = 11)

Demographic and clinical characteristics on admission

Male no. (%) 89 (71.2) 16 (64.0) 73 (73.7) 0.333 4 (40.0) 0.030 4 (36.4) -
Age years 68.0 (65.8 70.0) 66.4 (62.3 70.5) 68.4 (66.0 70.7) 0,092 59.0 (49.2 68.8) 0.010 34 (32 41) -
2
BMI kg/m 30.5 (29.2 31.7) 30.0 (27.2 32.8) 30.5 (29.2 31.9) 0,407 28.4 (25.7 31.0) 0.104 20.6 (19.6 22.9) -
Hypertension no. (%) 70 (56.5) 13 (52.0) 57 (57.6) 0.615 4 (40.0) 0.314 0 (0) -
Diabetes no. (%) 45 (36.3) 8 (32.0) 37 (37.3) 0.618 4 (40.0) 0.815 0 (0) -
Cancer no. (%) 21 (16.9) 5 (20.0) 16 (16.1) 0.648 1 (10.0) 0.562 0 (0) -

Laboratory findings
Lymphocytes /mm3 921 (750 1091) 807 (689 956) 931 (734 1128) 0,363 1393 (1014 1771) <0.001 - 1500 4000
Monocytes /mm3 506 (285 640) 540 (220 900) 516 (436 595) 0.406 351 (273 430) 0.103 - 200 800
CRP mg/L 115.1 (100.3 129.8) 146.8 (111.8 181.8) 110.2 (93.8 126.7) 0,276 67.8 (47 109) 0.070 - <5
C3 g/L 1.3 (1.3 1.4) 1.3 (1.2 1.4) 1.3 (1.3 1.4) 0,429 - - - 0.81 1.57
C4 g/L 0.4 (0.3 0.4) 0.3 (0.2 0.3) 0.4 (0.4 0.5) <0.001 - - - 0.13 0.39
CH50 UI/mL 74.6 (71.6 77.5) 75.2 (66.7 83.8) 74.0 (71.8 76.2) 0,202 - - - 41.7 95.1
Serum ferritin mg/mL 1520.7 (1227.3 1814.2) 3279.2 (1860.7 4697.8) 1338.8 (1101.1 1576.6) 0,023 590.7 (29.7 1151.6) 0.070 - 8 252
D-Dimers - ng/mL 2024.6 (1609.7 2439.4) 2787.3 (1899.9 3674.8) 1828.0 (1365.6 2290.4) 0,001 1388.8 (318.1 2459.4) 0,081 - < 500

B. Bonnet et al. / EBioMedicine 73 (2021) 103622

IL-6 pg/mL 243.7 (3.3 484.0) 837.3 (265.6 1940.1) 93.8 (9.8 197.3) <0.001 12.5 (9.7 15.3) <0.001 1.6 (0.5 3.7) <2.4
CXCL8 pg/mL 46.8 (29.8 63.8) 81.2 (27.6 134.7) 38.1 (22.0 54.3) <0.001 23.2 (15.4 31.0) <0.001 3.7 (2.7 4.7) <7.8
IFN-a pg/mL 8.4 (2.9 13.8) 17.7 (6.2 29.3) 7.4 (1.3 13.5) 0.020 8.8 (1.4 16.2) 0.217 1.2 (0.1 2.2) <4.4
IL-10 pg/mL 6.1 (3.9 8.3) 11.3 (1.6 21.0) 4.8 (3.7 6.0) 0.012 2.5 (1.5 3.6) 0.004 0.8 (0.2 1.4) <1.4
IL1Ra pg/mL 552.0 (386.9 717.3) 1284.1 (615.2 1953.1) 492.3 (332.7 652.0) 0.008 86.6 (66.1 107.1) 0.011 - -
mHLA-DR pg/mL 11600 (10436 12764) 5926 (5028 6824) 11772 (10468 13076) 0.033 21566 (18004 25128) 0.010 44544 (26884 62203) >15000

During hospital stay

Time from symptoms to unit 9.0 (4.7 13.4) 10.6 (6.1 15.0) 8.6 (4.3 13.0) 0,428 7.9 (5.6 10.2) <0.001 - -
admission day
SAPSII score 36.6 (34.5 38.6) 41.4 (36.2 46.5) 35.3 (33.2 37.5) 0,026 - - - -
Nosocomial infection no. 38 (30.6) 12 (48.0) 26 (26.3) 0.035 0 (0) 0.039 - -
(%)
Including septic shock no. 20 (16.1) 5 (20.0) 15 (15.2) 0.556 - - - -
(%)
Length of stay days 9.6 (7.8 11.4) 13.2 (6.3 20.1) 8.6 (7.2 10.1) 0.007 5.7 (2.1 9.3) 0.030 - -
Death in hospital no. (%) 35 (28.2) 10 (40.0) 25 (25.3) 0.090 0 (0.0) 0.060 - -
Discharge from hospital 91 (73.4) 15 (60.0) 76(76.8) 0.090 10 (100.0) 0.060 - -
no. (%)
Data are expressed as mean [IC95] or percentages (%). ICU: Intensive care unit; IDU: Infectious disease unit; BMI: Body mass index; CRP: C-reactive protein; C3, C4, CH50: Complement fractions; ARDS: Acute respiratory distress syndrome;
SAPS II: Simplified Acute Physiology Score II.
 B. Bonnet et al. / EBioMedicine 73 (2021) 103622
Fig. 1. Flow chart of the study.
higher increase in serum ferritin and CRP and a statistically signifi-
cant decrease in lymphocyte cell counts (Table 1). Thirty seven
patients (30.6%) in the ICU as against none in the IDU developed a
microbiologically-proven secondary infection, including 20 septic
shocks (16.1%). Finally, 35 patients (28.2%) died in the ICU whereas
all IDU patients recovered and were discharged (Table 1). Patient
demographic characteristics on admission were not statistically dif-
ferent between the two waves of hospitalization. However, patients
from Wave 1 were in a more severe condition than Wave 2 patients,
as shown by higher CRP, D-Dimers and serum ferritin, associated
with higher SAPSII scores, a longer length of stay in intensive care
and the occurrence of statistically more secondary infections
(Table 1). Medication and medical care administered to all ICU
patients are given in Table S2. Overall mortality decreased in Wave 2,
but not to a degree of significance.
3.2. COVID-19-induced inflammation at different times of ICU
hospitalization
Upon admission, plasma concentrations of IL-6 and CXCL8 were
statistically higher in patients who were primarily admitted to the
ICU than in those admitted to a conventional IDU ward, whose con-
centrations were nevertheless statistically higher than those of the
healthy controls (Table 1). In addition, changes in the management of
ICU patients from June 2020 resulted in several modifications to pro-
inflammatory cytokines. Specifically, Wave 1 patients, who were pri-
marily admitted to the ICU up to March 2020, had a significant 8.9-
fold increase in mean IL-6 values (837.3 pg/mL) compared to ICU
patients from Wave 2 (93.8 pg/mL) (table 1). Also, mean CXCL8 con-
centrations were significantly 2.1-fold higher in Wave 1 ICU patients
(81.2 pg/mL) than in Wave 2 ICU patients (38.1 pg/mL). No
5
statistically significant difference in IFN-a values was observed
between IDU and ICU patients, but the initial mean IFN-a level of
Wave 1 ICU patients was statistically higher than in Wave 2 ICU
patients (Table 1).
Interestingly, 3.2% (n = 4/124) of ICU patients developed a condi-
tion compatible with CSS, whose threshold was defined as the mean
of plasma cytokine concentrations + 2 SD by Mudd et al. [24]. Of these
patients, 3 were from Wave 1 (12%, n = 3/25) and only one from Wave
2 (1%, n = 1/99) (p = 0.01, Fisher’s exact test) (Table S3). All but one of
these patients died. None of these patients had a ferritin level greater
than 4420 ng/mL (data not shown). The only survivor had been
treated with tocilizumab, which produced a progressive decline in
plasma IL-6 (data not shown). Finally, plasma IL-6 and the PaO2/FiO2
ratio at admission were well inversely associated in Wave 1 patients
(r = -0.49) but weakly inversely correlated in Wave 2 patients (r =
-0.16, Fig. 2a). Plasma IL-6 (p = 0.02, Mann Whitney U test), but not
CXCL8 (p = 0.2567, Mann Whitney U test) values at admission were
statistically higher in ICU COVID-19 patients with PaO2/FiO2 ratio
less than 100 compared to others (Figure S2).
We re-analysed the cytokine results on admission no longer by
grouping the patients by wave but depending on whether they had
received corticosteroids or not. This confirmed that blood levels of
pro-inflammatory cytokines were statistically lower in patients who
had received corticosteroids (Figure S3).
We next focused on hospitalized ICU patients, monitoring their
cytokine profile over time according to disease outcome, fatal or not.
The mean IL-6 level of Wave 2 survivor patients hospitalized in the
ICU dropped from 33.4 pg/mL at admission to 11.3 pg/mL at dis-
charge, which represents a statistically significant decrease from
baseline (p = 0.003, Mann Whitney U test), and reached IDU values
(dotted grey line in Fig. 2b). The mean CXCL8 concentration of
6 B. Bonnet et al. / EBioMedicine 73 (2021) 103622

Fig. 2. Plasma pro- and anti-inflammatory cytokine levels and monocyte dysregulation are associated in COVID-19 patients with pulmonary involvement, disease severity and mor-
tality. (a) Spearman correlation of plasma levels of IL-6 or mHLA-DR and PaO2/FiO2 ratios on admission in COVID-19 individuals. The black full line and the grey full line represent
the best fit linear relationship of data collected during Wave 1 and Wave 2, respectively. The black dotted lines represent the IC95. Evolution of plasma (b) pro-inflammatory cyto-
kine levels or (c) immunosuppression markers over time during ICU hospitalization measured at baseline (D0-3), on D7-10 and until discharge of the patient for recovery (Endpoint)
in alive (blue diamond) and deceased patients (red diamond). Grey diamonds represent patients deceased before D7 (n = 4). Grey dotted lines represent means of cytokines in IDU
patients at baseline. Each value represents the mean § SD. Only statistically significant results were indicated (¨ p < 0.05, ¨¨p < 0.01, versus baseline, Wilcoxon matched pairs test,
**p < 0.01, ***p < 0.001, alive patients vs deceased patients, Mann Whitney U test). Number of cases for each time point, S: Survivors, NS: Non- survivors. Time D0-3 70(S), 21(NS);
D7-10 23(S), 17(NS); Endpoint 18(S). PaO2: Patient's oxygen in arterial blood; FiO2: Fraction of the oxygen in the inspired air.

survivor patients was 29.1 pg/mL at admission, 38.2 pg/mL at day 7-
10, and 19.0 pg/mL at discharge: it decreased over time and was
below IDU values at ICU discharge (dotted grey line in Fig. 2b). We
chose to focus on the patients of Wave 2 because there were
markedly more of them. Changes in CXCL8 levels over time had a
fairly close profile in Wave 1 patients, but IL-6 levels remained stable
over time (Figure S4). We divided non-survivors into two groups, one
in which patients died less than 5 days after ICU entry and who there-
fore had no second sample taken on days 7-10, and the other for
deaths occurring later. Remarkably, the 4 patients who died early
had much higher plasma IL-6 and CXCL8 concentrations at baseline
than other deceased ICU patients (Fig. 2b). The other non-survivor
patients had statistically higher plasma IL-6 and CXCL8 concentra-
tions (mean IL-6 = 52.8 pg/mL, mean CXCL8 = 33.0 pg/mL) at baseline
(p = 0.008 and p = 0.009, respectively, Mann Whitney U test) and on
days 7-10 (p < 0.0001 and p = 0.006, respectively,
Mann Whitney U test) than survivor patients, with a statistically sig-
nificant increase over time until death (p = 0.0108 for IL-6 and
p = 0,0007 for CXCL8, Wilcoxon matched pairs test, Fig. 2b). The IFN-
a levels of survivors decreased over time, starting at 5.6 pg/mL at
admission and reaching 0.1 pg/mL at discharge, below IDU values
(dotted grey line in Fig. 2b). The initial high IFN-a levels of non-survi-
vor patients decreased over time to below-normal values at death
(Fig. 2b). Finally, plasma IL-6 concentrations and PaO2/FiO2 ratio
measured at all time points were significantly inversely correlated
(r = -0.25, data not shown).
3.3. Severe COVID-19 diseases showed biological signs of
immunosuppression
Upon admission, plasma concentrations of IL-10 were statistically
higher in patients who were admitted to the ICU than those of
patients admitted to conventional units, which in turn were statisti-
cally higher than those of healthy controls (Table 1). In addition,
Wave 1 patients had a 2.4-fold significant increase in mean IL-10 con-
centrations (11.3 pg/mL) compared to ICU patients from Wave 2 (4.8
pg/mL) (Table 1). Notably, plasma IL-1Ra levels at admission were
also statistically higher in ICU patients than in IDU patients, and
Wave 1 ICU patients had a 2.6-fold significant increase in mean
 B. Bonnet et al. / EBioMedicine 73 (2021) 103622 7

Fig. 3. Severe COVID-19 patients had both cytokine inflammation and monocyte dysregulation. (a) Spearman correlation of HLA-DR expression on monocytes and plasma IL-6 lev-
els (D0-3, D7-10 and Endpoint) in Wave 2 ICU COVID-19 individuals. The full line represents the best fit linear relationship of data. (b) Evolution of IL-6/mHLA-DR ratio over time
during ICU hospitalization measured at baseline (D0-3), at D7-10 and until discharge of the patient for recovery (Endpoint) in alive (blue diamond) and deceased patients (red dia-
mond). Grey diamonds represent patients deceased before D7 (n = 4). Grey dotted lines represent the mean of IL-6/mHLA-DR ratio in IDU patients at baseline. Each value represents
the mean § SD. Only statistically significant results were indicated (¨¨p < 0.01 versus baseline, Wilcoxon matched pairs test, ***p < 0.001, alive patients vs deceased patients,
Mann Whitney U test). Number of cases for each time point, S: Survivors, NS: non survivors. Time D0-3 70(S), 20(NS); D7-10 23(S), 16(NS); Endpoint 18(S).

concentrations of this cytokine (1284.1 pg/mL) compared to ICU
patients from Wave 2 (492.3 pg/mL) (Table 1).
In contrast, mHLA-DR expression was dramatically lower in COVID-
19 patients upon admission than in healthy controls and was statistically
decreased by two fold in ICU patients compared to IDU patients (Table 1).
In addition, ICU patients from Wave 1 had a statistically lower expression
of mHLA-DR than Wave 2 patients (Table 1). Finally, mHLA-DR expres-
sion and PaO2/FiO2 ratio measured at admission were significantly corre-
lated (Fig. 2a). Plasma IL-10 values at admission were non-significantly
higher (p = 0.1043, Mann Whitney U test) but mHLA-DR expression
significantly decreased (p < 0.001, Mann Whitney U test) in ICU COVID-
19 patients with PaO2/FiO2 ratio less than 100 than in other patients
(Figure S2).
Among ICU hospitalized patients, IL-10 concentrations in survi-
vors decreased over time (Fig. 2c), starting at 3.6 pg/mL at admission
and reaching 2.0 pg/mL at discharge (p = 0,0407,
Mann Whitney U test), close to IDU values (dotted grey line in
Fig. 2c). Conversely, non-survivors had statistically higher plasma IL-
10 concentrations at baseline (mean IL-10 = 8.6 pg/mL) than did sur-
vivors, which remained higher on days 7-10 but without change over
time (Fig. 2c). There was no difference in IL-10 values at admission
between patients who died early and those who died later. Data from
Wave 1 yielded the same observations (Figure S4).
The mean mHLA-DR expression of survivor patients hospitalized
in the ICU was 12,414 Ab/monocyte at admission, 8,783 Ab/monocyte
on days 7-10, and 17,069 Ab/monocyte at discharge, with an increase
over time (p = 0.0081, Wilcoxon matched pairs test Fig. 2c). Non-sur-
vivors had significantly down-regulated mHLA-DR expression at
baseline (mean = 9644 Ab/monocytes) compared to survivors (p =
0.0171, Mann Whitney U test). There was no difference in mHLA-DR
expression on admission between patients who died early and those
who died later. The difference between survivors and non-survivors
was significantly accentuated on days 7-10 (p < 0.0001,
Mann Whitney U test), with expression of mHLA-DR in the latter
group decreasing to 4,325 Ab/monocyte on average (p = 0.0004, Wil-
coxon matched pairs test Fig. 2c). Finally, expression of mHLA-DR
and PaO2/FiO2 ratio measured at all time points were significantly
correlated (r = 0.33, data not shown).
3.4. Severe COVID-19 patients had both cytokine inflammation and
monocyte dysregulation
In Wave 2 COVID-19 ICU patients, blood mHLA-DR expression and
plasma IL-6 levels were significantly inversely well correlated at all
time points (Fig. 3a). To simultaneously analyse both facets of the
immune response, we performed an IL-6/mHLA-DR*103 ratio
analysis. Survivor ratio was 3.4 at admission, 4.1 on days 7-10, and
0.89 at discharge, with a decrease over time to reach IDU values at
ICU discharge (dotted grey line in Fig. 3b). Remarkably, the 4 patients
who died early had a much higher IL-6/mHLA-DR*103 ratio at base-
line (ratio of 185.0) than other deceased ICU patients (Fig. 3b). The
other non-survivor patients had a statistically higher IL-6/mHLA-
DR*103 ratio at baseline (ratio of 6.6) and on days 7-10 (ratio of
110.5) than survivor patients, with a very significant increase over
time until death (Fig. 3b).
The ROC curve analysis identified the optimal cut-off values of
baseline and D7 mHLA-DR, IL-6 or IL-6/mHLA-DR*103 parameters for
prediction of fatal COVID-19 (Fig. 4, Table 2). These optimal cut off
points showed high negative predictive values (NPV) for the different
parameters ranging from 86% to 100%. We also determined for the
different parameters the thresholds that yielded positive predictive
values (PPV) of 100% in order to identify patients at high risk of death
(Table 2).
3.5. Inflammation and monocyte dysregulation in ICU COVID-19
patients with secondary infections
Secondary infections occurred in 26 out of 99 Wave 2-COVID-19
ICU patients. The microbiological data of those infections are given in
supplemental Table S4. On admission, COVID-19 ICU patients who
had experienced a secondary infection during their stay were signifi-
cantly more inflammatory than those who had not, as evidenced by a
10-fold increase in plasma IL-6 during both waves (Fig. 5a). At the
same time, these patients also had significantly down-regulated
mHLA-DR expression during Wave 2, from 12,880 to 8,726 molecules
per monocyte (Fig. 5b). Secondary infected patients had statistically
increased IL-6/mHLA-DR*103 ratio values compared to non-second-
ary infected patients in both waves, resulting in 53-fold change and
9-fold change increases, respectively, for Wave 1 and Wave 2
(Fig. 5c). In Wave 2, 13 patients out of 26 who experienced a second-
ary infection developed a life-threatening infection and died
(Table S4).
The ROC curve analysis identified the optimal cut-off values of
mHLA-DR, IL-6 or IL-6/mHLA-DR*103 parameters for prediction of
the occurrence of secondary infection (Figure S5, Table S5). These
optimal cut off points showed PPV of 100% at baseline for the differ-
ent parameters and PPV ranging from 64% to 100% on days 7-10.
We divided the ICU patients with a secondary infection into two
groups, one with early infections occurring within less than 3 days
after ICU admission and another for infections that occurred more
than 4 days after admission. Remarkably, IL-6 plasma levels in
patients with early secondary infections (mean IL-6 = 558.2 pg/mL)
8 B. Bonnet et al. / EBioMedicine 73 (2021) 103622

Fig. 4. Receiver operating characteristic (ROC) curves predicting unfavorable outcome of COVID-19 in ICU patients. Receiver operating characteristic (ROC) curves of (a) mHLA-DR,
(b) plasma IL-6 and (c) IL-6/mHLA-DR ratio, obtained at baseline (red) and at D7-10 (blue), predicting unfavorable outcome (death) of COVID-19 in ICU patients. Area under the ROC
curve with 95% CI are indicated for each parameter.

Table 2
Cut-off values for mHLA-DR, plasma IL-6 and IL-6/mHLA-DR ratio analysed at D0 and D7-10 according to Youden’s index or
maximum PPV or NPV predicting unfavourable outcome (death).

Laboratory tests Optimal cut-off Sp - % (CI95) Se - % (CI95) Npv - % Ppv - %

Youden’s index
mHLA-DR (D0-3) Ab/c 11312.5 54 (43 65) 74 (57 91) 87 34
mHLA-DR (D7-10) Ab/c 4672.5 86 (71 - 96) 75 (50 94) 86 75
IL-6 (D0-3) pg/mL 19.1 58 (47 70) 91 (78 100) 95 41
IL-6 (D7-10) - pg/mL 14.3 57 (39 75) 100 (100 100) 100 57
IL-6 / mHLA-DR *10^3 (D0-3) 1.4 56 (44 66) 96 (87 100) 98 41
IL-6 / mHLA-DR *10^3 (D7-10) 2.7 68 (50 86) 100 (100 100) 100 64
Maximum PPV = 100%
mHLA-DR (D0-3) - Ab/c 2795.5 100 4.3 (0 13) 77 100
mHLA-DR (D7-10) - Ab/c 1361.0 100 6.3 (0 19) 65 100
IL-6 (D0-3) - pg/mL 226.7 100 13 (0 30) 78 100
IL-6 (D7-10) - pg/mL 236.1 100 38 (13 63) 74 100
IL-6 / mHLA-DR *10^3 (D0-3) 18.1 100 22 (4 40) 80 100
IL-6 / mHLA-DR *10^3 (D7-10) 49.6 100 38 (13 63) 74 100
Maximum NPV = 100%
mHLA-DR (D0-3) Ab/c 19081.0 17 (8 26) 100 100 28
mHLA-DR (D7-10) Ab/c 12615.0 25 (10 43) 100 100 43
Sp: Specificity; Se: Sensitivity; NPV: Negative predictive value; PPV: Positive predictive value, CI95: Confidence interval 95%

were not statistically different from those in patients with later infec-
tions (mean IL-6 = 53.3 pg/mL) (Fig. 5d). A notable exception was one
Wave 2 patient who experienced a cytokine storm with IL-6 values at
admission of 4981 pg/mL, which was 88 times higher than the
median assay of the early infection group to which he belonged. Con-
versely, the levels of mHLA-DR were statistically different between
the two groups of patients, with a two-fold downregulation in those
with early secondary infections (Fig. 5e). Specifically, 26 ICU patients
had at least one severe secondary infection in Wave 2. Of the 9 who
developed early infections, all had an initial mHLA-DR lower than
9,000 Ab/monocyte (Table S6). Conversely, 10 out of 17 (59.0%) other
patients who developed later infections after admission had initial
mHLA-DR expression greater than this threshold. Likewise, we
observed a difference in the IL-6/mHLA-DR ratio between the two
groups, with about a 2-fold increase in the median in patients with
early infection (Fig. 5f). In patients who developed infections 4 days
after ICU admission, mHLA-DR expression recorded on days 7-10, ie
near the onset of secondary infections, was statistically lower than
that at admission and reached the mHLA-DR value obtained for ICU
patients who presented early infections on days 0-3 (Fig. 5g). The
lack of data on mHLA-DRs expression during Wave 1 prevented us
from distinguishing between the relative impacts of IL-6 and mHLA-
DR on the occurrence of secondary infections. However, it is impor-
tant to emphasize that, as for the patient with a cytokine storm
described above, all patients with pro-inflammatory cytokine storm
syndrome (CSS) in Wave 1 had secondary infections. There is there-
fore an over-representation of infections of this type in the CSS ICU
patient group compared to non-CSS ICU patients (3/3 = 100% vs 9/
22 = 40.9%).
4. Discussion
Patients with severe forms of COVID-19 have mortality rates in
the ICU which remain high to this day. They most often die of respira-
tory failure after the development of ARDS. However, other factors
may be associated with an increased risk of death, including second-
ary infections. Based on what we knew and practised in the context
of sepsis, we hypothesized that severe COVID-19 patients were likely
to suffer from a complex immune dysfunction combining inflamma-
tion and immunosuppression.
In our series, the plasma IL-6 and CXCL8 levels in the different
severity groups of COVID-19 patients were statistically different on
admission. This is clear evidence that severe ICU COVID-19 patients
were experiencing inflammation. The cytokine results were also
inversely correlated with the PaO2/FiO2 ratio, attesting to a link
between the degree of inflammation and the pulmonary involvement
of ARDS. Thus, in our series, as in other previous studies, the degree of
initial inflammation appears to be predictive of the severity of
COVID-19, both in terms of the severity of the lung injury and of the
risk of death [3,10,12,25]. However, in agreement with recent data
 B. Bonnet et al. / EBioMedicine 73 (2021) 103622 9

Fig. 5. Cytokine inflammation and monocyte dysregulation at admission are more marked in COVID-19 ICU patients who will subsequently suffer from secondary infections. (a)
Plasma IL-6 levels, (b) Monocyte HLA-DR membrane expression and (c) IL-6/mHLA-DR ratio were analysed at admission in ICU patients from Wave 1 (black bars) and Wave 2 (grey
bars) according to the occurrence of secondary infections acquired during hospitalization. Bar graphs represent mean § standard deviation (Statistical comparison by Mann-Whit-
ney U test). Wave 2 ICU patients at baseline (d) plasma IL-6 levels, (e) monocyte HLA-DR membrane expression and (f) IL-6/mHLA-DR ratio were detailed according to the onset of
secondary infection, defined as early infection (D0-3) or late infection (> D4). Each dot represents an individual value, and mean § standard deviation are shown (Statistical compar-
ison by Mann-Whitney U test). Dotted line in (e) represents mHLA-DR threshold predicting early secondary infection occurrence. (g) Monocyte membrane expression of human leu-
cocyte antigen antigen D-related (mHLA-DR) results from individual patients with late secondary infection, represented as dots in the figure, with lines connecting baseline mHLA-
DR expression and D7-10 mHLA-DR expression. The time of onset of infection is shown by the grey area in the figure. Horizontal grey dotted lines represent the mean of mHLA-DR
expression at D0-3 in ICU patients with early infection. Horizontal red dotted lines represents mHLA-DR threshold predicting early secondary infection occurrence (comparison ver-
sus baseline, Wilcoxon matched pairs test).

from the literature and with our own (not shown), it is noteworthy
that the mean values of these cytokine levels were much lower than
in other pathological settings, such as bacterial septic shocks, other
causes of ARDS or injections of CAR T cells, and quite close to levels
induced by classical viral respiratory infections like influenza
[15 17,24]. In contrast to prevailing hypotheses about the patho-
physiology of COVID-19 disease very few patients in our series pre-
sented cytokine profiles indicative of CSS. In the absence of any
internationally recognized criterion we set the threshold according
to the method recently described by Mudd et al., which indicated
that only 4 of our COVID-19 patients had CSS [24], all of whom were
hospitalized in the ICU and none in the IDU. Although cases of CSS
were rare in our patient series they nevertheless made up a severe
subgroup with a very poor prognosis, since 3 out of the 4 patients
who met the criterion on admission died during hospitalization, a
mortality rate of 75% compared to an overall mortality in ICU patients
of 25%. Note that for the 3 patients for whom we had a ferritin assay,
none reached the threshold of 4420 ng / mL, previously used for
evoking a macrophage activation syndrome during COVID-19 [21].
Another interesting point among ICU patients was the dramatic
decrease in proinflammatory cytokines measured during Wave 2
compared to Wave 1. In particular, 12% of patients met the criteria
for CSS during Wave 1 as against 1% during Wave 2. These results
show that CSS, and hence hyperinflammation, was not that rare dur-
ing Wave 1 but much more so during Wave 2. They should be put in
perspective with the significant change in management practices
introduced in our teaching hospital between the two waves. Follow-
ing the publication in July 2020 of the RECOVERY trial, the great
majority of patients in Wave 2 received dexamethasone (89%)
compared to a small minority in Wave 1 who had received corti-
costeroids (5/25, ie 20% of patients receiving between 10 and
60 mg of prednisone per day) [23]. One of the main known phar-
macological mechanisms of the action of glucocorticoids is to
inhibit the synthesis of pro-inflammatory cytokines [26]. It makes
sense to think that the decrease in cytokine levels that we saw in
Wave 2 was due to the almost systematic use of this molecule.
However, to our knowledge, we are the first to demonstrate this
in comparative data [27].
10 B. Bonnet et al. / EBioMedicine 73 (2021) 103622
To assess whether COVID-19 induced monocyte dysregulation, we
required a test that was both able to test a key functionality of the
immune system and routinely available. Our choice quickly fell on
the membrane expression of HLA-DR on blood monocytes. Low
mHLA-DR has become one of the best markers of monocyte immuno-
suppressive phenotype in various diseases such as cancers and sepsis
[22,28 31]. The scientific rationale for these findings, from a funda-
mental immunological point of view, is that HLA-DR is the main anti-
gen-presenting molecule for helper T cells on the surface of
monocytes, macrophages and dendritic cells. Thus, it is assumed that
the decrease in mHLA-DR on the surface of blood monocytes is a
reflection of a reduced overall capacity for presenting antigens and
therefore for inducing adaptative immune responses. The anomalies
of the myeloid compartment induced by COVID-19 are being gradu-
ally documented, in particular those concerning monocytes, with
early expansion and activation of blood classical CD14+ monocyte
expressing HLA-DR++ being seen in moderate COVID-19 patients and
conversely an accumulation of HLA-DRlo monocytes in severe
patients [11,23,24,26,27]. All these data confirm the potential interest
of the marker of monocyte immunosuppressive phenotype that we
chose [32 34].
In our series, on the basis of recognized interpretation thresholds
from the literature for the analysis of mHLA-DR expression, it can be
considered that IDU patients with mild forms, despite having lower
values than healthy controls, are not immunosuppressed on admis-
sion (mean HLA-DR = 21566 Ab/cells) unlike ICU patients, who clearly
are [20,31,35,36]. Hence, as previously reported, decreased mHLA-DR
was a significant predictor of COVID-19 disease severity [24,37,38]. In
our study, levels of IL-10, one of the main anti-inflammatory cyto-
kines, were statistically higher in ICU patients than in IDU patients
and healthy controls. In addition, concentrations of IL-1Ra, a cytokine
of the acute phase of inflammation which plays an anti-inflammatory
role of counter-regulation, in particular at the level of monocytes
(both target and producer of the molecule), were also statistically
higher in ICU patients than in IDU patients. The profile of secretion of
these two molecules therefore confirmed, in agreement with mHLA-
DR data, the presence of immunosuppression in the most severe
patients.
Comparison of the results of ICU patients between the two waves
showed that patients in Wave 1 expressed monocyte mHLA-DR sig-
nificantly less than those in Wave 2. On the basis of the same criteria
as before, Wave 1 patients exhibited severe monocyte dysregulation,
on the verge of immunoparalysis (mean HLA-DR = 5926 Ab/cells)
[31,35], unlike Wave 2 patients, who were in a state of simple mono-
cyte dysregulation (mean HLA-DR = 11772 Ab/cell). Here again, IL-10
and IL-1Ra levels were in close agreement with mHLA-DR results,
with Wave 1 ICU patients showing higher concentrations of both
immunosuppressive cytokines. One could suspect again that system-
atic treatment with dexamethasone played a role [39,40].
Comparison of the mHLA-DR data in the different groups of
COVID-19 patients on ICU admission according to mortality showed
that the expression of the marker was inversely proportional to the
severity of the disease. The time-dependent evolution of mHLA-DR
should be emphasized because in the two groups of patients, survi-
vors and non-survivors, a statistically significant decrease in values
was observed on days 7-10 compared to baseline. However, the dif-
ference in values between the two groups increased statistically,
with surviving patients remaining in the zone of simple monocyte
dysregulation while those in the non-survivor group became severely
immunosuppressed [31,35]. Subsequently, in surviving patients, a
statistically significant increase in measurements was observed
before ICU discharge, with values returning to normal, thereby indi-
cating the disappearance of monocyte dysregulation. It can thus be
considered that the evolution of mHLA-DR follows a V trend curve, as
recently reported, with a nadir probably between days 7 and 10 after
ICU admission when immunosuppression is therefore probably at its
greatest [41,42]. All these data show, for the first time to our knowl-
edge, that the quantitative expression of HLA-DR on monocytes is a
predictive marker of mortality in COVID-19 patients. They are to be
compared with those of a previous study which found a decrease in
the number of CD14+ HLA-DR+ monocytes in patients who died of
COVID-19 compared to survivors [33]. In addition, mHLA-DR quanti-
tative expression during hospitalization was also significantly corre-
lated with the PaO2/FiO2 ratio thereby indicating, as previously
described, a relation between the degree of monocyte dysregulation
and the pulmonary involvement of ARDS [21]. Again, it is noteworthy
that the IL-10 results are in agreement with those of mHLA-DR, nota-
bly with IL-10 levels being very significantly greater in the non-survi-
vor group than in survivor patients. Thus, during severe COVID-19
infections, we found an immunosuppressive profile associating IL-10
production and a decrease in mHLA-DR the same as that already
observed in septic shocks [43]. We could not evaluate it for reasons
of simplicity and effectiveness contrary to our routine care approach,
but we can hypothesize, given the data in the literature, that this
result probably reflects a more overall myeloid (granulocytes, macro-
phages, dendritic cells) and lymphoid dysregulation which would be
interesting to investigate in later studies.
Interestingly, we showed that IL-6 and mHLA-DR were well corre-
lated in ICU patients, in an inversely proportional way. This enables
us to assert that inflammation and monocyte dysregulation are
indeed phenomena occurring concomitantly in patients with severe
forms of COVID-19 and therefore confirm our initial hypothesis. Con-
sequently, the use of the IL-6/mHLA-DR ratio could be of interest to
simultaneously analyse the two aspects of the immune response in
the same patient. In ICU patients, the ratio on admission was signifi-
cantly higher in the non-survivor group than in the survivor group,
and the difference was even more marked on days 7-10, confirming
that the inflamed/immunocompromised dual state is associated with
poor prognosis. The analysis of ROC curves for IL-6 concentrations,
HLA-DR expression, and IL-6/mHLA-DR ratio on admission showed
that IL-6/mHLA-DR ratio was the more reliable cut-off for predicting
fatal outcome. Thus, we could propose the use of IL-6/mHLA-DR ratio
in current clinical practice, with a threshold of 18, as in our series, to
obtain a PPV of the risk of death of 100%. To our knowledge, this is
the first report to demonstrate that IL-6/mHLA-DR ratio is a valuable
marker for predicting death in COVID-19. This interesting result
needs to be confirmed soon using the proposed threshold prospec-
tively in studies involving a greater number of patients.
These results should be compared with those of recent studies
suggesting that immune dysregulation during COVID-19 with early
and prolonged immune system activation can result in cellular
exhaustion [44]. More precisely, it has been proposed that severe
COVID-19 patients suffered from a defective antigen-presentation,
evidenced by a decrease in mHLA-DR and which, associated with
lymphopenia, led to defective function of lymphoid cells, whereas
monocytes remained potent for the production of TNFa and IL-6.
Moreover, the involvement of IL-6 in the mHLA-DR decrease has
been demonstrated in part by the fact that an IL-6 blocker, Tocilizu-
mab, partially rescued this downregulation in vitro [21,45]. Our
results could be considered as consistent with this observation. How-
ever, we cannot rule out a role for IL-10 as well since levels of the
cytokine were also higher in our severe patients, and previous publi-
cations have shown its involvement in the internalization of HLA-DR
molecules in monocytes in the context of septic shock [46]. Another
factor that could contribute to the decrease in HLA-DR on monocytes
was the involvement of neutrophilic granulocytes. The role of the
activation of these cells in COVID-19, which has been reported by
various teams, seems to be corroborated in our study by the increase
in CXCL8 levels in severe forms. In particular, the secretion of neutro-
philic elastase is associated with the severe forms of the disease
[47,48]. However, once released this elastase has the property of lys-
ing nearby HLA-DR molecules [49]. In our series, we observed an
 B. Bonnet et al. / EBioMedicine 73 (2021) 103622
increase in this marker in our most severe patients and an inverse
correlation with the expression of HLA-DR (data not shown). How-
ever, these preliminary results need to be confirmed in other studies.
To demonstrate that the changes in our markers in severe COVID-
19 forms, especially the decrease in HLA-DR on monocytes, were rel-
evant functional indicators of immunoparesis of the immune system,
we decided to correlate the occurrence of serious secondary infec-
tions over time with the expression of the markers to see if they
were predictive of a risk of secondary infections in the short term.
Although relatively unknown, it is very common for COVID-19
patients to develop secondary infections during ICU hospitalizations
with several studies reporting superinfection rates higher than 50%
[8,9]. The occurrence of secondary infections is also recognized as a
clinical predictor of fatal outcome in COVID-19 cases [50]. In our
study, we can see that 52% of total deaths from Wave 2 were directly
attributable to severe secondary infections, which makes these infec-
tions the main cause of death in our patients. On admission, IL-6 con-
centrations, mHLA-DR expression, and IL-6/HLA-DR ratio were all
statistically higher in patients who later developed a secondary infec-
tion at some point during their hospitalization. Conversely, when we
compared the two groups of patients with early or later infections,
the IL-6 values were at levels not statistically different. Interestingly,
mHLA-DR expression was on the other hand statistically lower in the
early infections group than in the group with later infections with a
threshold differentiating between the two groups set empirically
(because unfortunately we did not have enough points to set a
threshold using interpretable ROC curves) at around 9000 Ab/mono-
cyte. Of note, the group of patients with later infections downregu-
lated mHLA-DR expression to less than this threshold on day 7, the
moment when they in turn developed secondary infections. These
results clearly demonstrate that the decrease in mHLA-DR expres-
sion, but not the slight increase in IL-6 levels, corresponds to an
immunosuppression state correlated with the risk of the rapid occur-
rence of secondary infections. Finally, we show, for the first time to
our knowledge, that the downregulation of HLA-DR on monocytes is
a predictive marker of early secondary infections in COVID-19
patients. With regard to IL-6, the only patient who presented a major
CSS during Wave 2 developed a very early secondary infection. Like-
wise, the 3 patients who had a cytokine storm during Wave 1 also
developed early secondary infections. Unfortunately, HLA-DR expres-
sion was measured in only one patient, who was severely immuno-
compromised (3422 Ab/monocyte). Thus, patients with COVID-19-
induced CSS in our study seem to be a subgroup at high risk for sec-
ondary infections. Cytokine storms therefore seem to be occurrences
that associate massive inflammation and severe immunosuppression.
4.1. Caveats and Limitations
Among the main limitations of this study was the relatively small
population size, in particular the healthy controls, IDU patients and
Wave 1 ICU groups. This was in part due to the health situation dur-
ing the first wave, when the partial saturation of our hospital created
difficulties in organizing the samples, and stocks of certain cytometry
reagents were in short supply. It should be noted that we have
recently analysed 18 additional IDU patients. Interestingly, they
exhibited an immune profile identical to that of the patients pre-
sented in this study (Figure S6). In particular, there was no significant
difference between the values obtained for the two IDU groups for all
parameters tested. These new patients were included from 11/17/
2020 to 05/02/2021, a period in France corresponding to the occur-
rence of the third epidemic wave (with the introduction into the ter-
ritory of the Alpha variant), and so we preferred not to pool their
results with those of the initial group to avoid introducing too much
heterogeneity between the patients. The results nevertheless give
reassuring arguments as to the validity of our results for IDU patients.
Another limitation of our study, which is related to our decision to
11
adopt a routine care approach, is that we assessed the degree of
immunosuppression of patients on only three markers (mainly
mHLA-DR) and did not perform complex multiparametric analyses to
assess more extensively all the immune cell populations involved in
the complex immune deregulation of COVID-19. In addition, direct
conclusions on the causality between disease severity and immuno-
logical profiles and the use of dexamethasone cannot be drawn from
our study due to its explorative nature. It would therefore be interest-
ing to try a functional approach to test whether the monocytes of
severe COVID-19 patients with downregulated mHLA-DR have
altered properties, for example in terms of phagocytosis or cytokine
synthesis in response to various stimuli, especially with regard to
their possible mechanistic role in disease course and severity. We
plan to test these last three points in an ancillary study that we will
carry out soon of new patients. Patient assignment to our cohort was
not random but exclusively based on the notion of hospitalization.
The introduction of bias due to this approach cannot be excluded as it
is possible that because of their profile certain patients were not hos-
pitalized in teaching hospitals or that particularly severe patients
died before they could be hospitalized. The fact that there are very
few intensive care beds in our region other than those at our hospital
appears to minimize this risk. We are unable to protect against the
risk of death before hospitalization and this is why we made mention
of it earlier in the text.
Finally, in the analysis of disease severity, we classified the
patients according to admission to ICU versus IDU wards, which
might differ from classifications used in other studies. In line with
numerous previous works, we observed a relationship between the
degree of increase in proinflammatory markers and disease severity.
Plasma sampling was performed at pre-defined time points depend-
ing on the day of hospital admission but it could be argued that dif-
ference in severity is attributable to longer disease duration.
Nevertheless, while this may have been theoretically a potential con-
founder, the time from disease onset to hospital admission was
recorded and showed no significant difference between the two ICU
groups. To limit the heterogeneity of the ICU patient group and to be
able to compare it with the IDU group, we decided to include for anal-
ysis only patients admitted directly to the ICU and those who were
admitted after prior admission to another department for a period <
3 days. As a consequence of our study’s pragmatic design, we per-
formed no correction for other potential confounders, such as comor-
bidity, medication or invasive mechanical ventilation use.
In the future, personalized medicine approaches to COVID-19 and
complete immunomonitoring simultaneously investigating both
sides of the immune response will undoubtedly be taken into account
to choose the most appropriate therapeutic interventions since they
can be completely opposite (for example anti-IL-6 antibodies versus
immune stimulants such as GM-CSF or IL-7) according to the patient's
profile [51,52]. Finally, owing to the use of simple routine care biolog-
ical markers, our approach responds at least in part to the problem by
making it possible to better characterize the profile of the immune
response of patients with severe COVID-19 in terms of inflammation
or immunosuppression. With the exception of a subgroup of very
severe patients who experienced a major cytokine storm, the great
majority of our hospitalized patients had moderate inflammation
associated with severe monocyte dysregulation, which is predictive
of the severity of the disease, its mortality and the risk of secondary
infections.
Contributors
BE, BB and BS conceptualized and designed the study. BB, JC, LF,
BE, EC, CD, LC, MA, MB, MV and LH conducted the investigations
(recruiting patients or conducting laboratory tests). BB and BE con-
ducted formal data analysis. BB and BE wrote the original first draft
of manuscript. All authors reviewed the manuscript and gave
12 B. Bonnet et al. / EBioMedicine 73 (2021) 103622
significant input. BB, BE, BS, and CD had access to and verified all
underlying data. BB ensures data curation. The final version of this
paper was reviewed and approved by all authors.
Data Sharing Statement
Individual participant data cannot be made available due to EU
Data Protection Regulations (GDPR). A limited and completely anony-
mized version of the dataset can be obtained upon request. Labora-
tory protocols will be available upon request. Enquiries should be
directed to bevrard@chu-clermontferrand.fr.
Declaration of Competing Interest
Dr. Bonnet reports non-financial support from THE BINDING SITE
GROUP LTD, non-financial support from DIASORIN SA, non-financial
support from Werfen, outside the submitted work. Prof. SOUWEINE
reports personal fees from MSD, non-financial support from TTM
BARD, personal fees from SANOFI, personal fees from LABORATOIRE
AGUETTANT, outside the submitted work. All other authors have no
conflicts of interest to disclose.
Acknowledgements
We thank the promoters of the CORIMUNO-19 project (INSERM-
Assistance Publique des Ho^ pitaux de Paris) for their help, the patients
of our study for being part of this cohort (screening with signature of
a consent) without being included in the associated trials (due to
competition with other studies in progress). We thank Xavier Daudet,
Estelle, Chapon, Charlene Pierson, Aure
lie Briançon, Brigitte Goutte,
Marlene Ravel, Julia Mercier, Marion Gaudard, Sole ne Revy, Maud
Junda and Fre de

ric Due

e for their immense assistance in the labora-
tory during patient material collection. Thank you to the entire staff
of the Department of Infectious Diseases and intensive care unit for
their feedback and scientific discussions. We thank the AFRRI associa-
tion, who kindly provided the additional doses of IL-1ra. This study
was not supported by any grant.
Supplementary materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.ebiom.2021.103622.
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